Hsp105α Suppresses Hsc70 Chaperone Activity by Inhibiting Hsc70 ATPase Activity*

Hsp105α is a mammalian member of the HSP105/110 family, a diverged subgroup of the HSP70 family. Hsp105α associates with Hsp70/Hsc70 as complexes in vivo and regulates the chaperone activity of Hsp70/Hsc70 negatively in vitro and in vivo. In this study, we examined the mechanisms by which Hsp105α regulates Hsc70 chaperone activity. Using a series of deletion mutants of Hsp105α and Hsc70, we found that the interaction between Hsp105α and Hsc70 was necessary for the suppression of Hsc70 chaperone activity by Hsp105α. Furthermore, Hsp105α and deletion mutants of Hsp105α that interacted with Hsc70 suppressed the ATPase activity of Hsc70, with the concomitant appearance of ATPase activity of Hsp105α. As the ATPase activity of Hsp70/Hsc70 is essential for the efficient folding of nonnative protein substrates, Hsp105α is suggested to regulate the substrate binding cycle of Hsp70/Hsc70 by inhibiting the ATPase activity of Hsp70/Hsc70, thereby functioning as a negative regulator of the Hsp70/Hsc70 chaperone system.

Hsp105␣ is a mammalian member of the HSP105/110 family, a diverged subgroup of the HSP70 family. Hsp105␣ associates with Hsp70/Hsc70 as complexes in vivo and regulates the chaperone activity of Hsp70/ Hsc70 negatively in vitro and in vivo. In this study, we examined the mechanisms by which Hsp105␣ regulates Hsc70 chaperone activity. Using a series of deletion mutants of Hsp105␣ and Hsc70, we found that the interaction between Hsp105␣ and Hsc70 was necessary for the suppression of Hsc70 chaperone activity by Hsp105␣. Furthermore, Hsp105␣ and deletion mutants of Hsp105␣ that interacted with Hsc70 suppressed the ATPase activity of Hsc70, with the concomitant appearance of ATPase activity of Hsp105␣. As the ATPase activity of Hsp70/Hsc70 is essential for the efficient folding of nonnative protein substrates, Hsp105␣ is suggested to regulate the substrate binding cycle of Hsp70/Hsc70 by inhibiting the ATPase activity of Hsp70/Hsc70, thereby functioning as a negative regulator of the Hsp70/Hsc70 chaperone system.
Hsp105␣ and Hsp105␤ are mammalian stress proteins that belong to the HSP105/110 family. Hsp105␣ is constitutively expressed and induced by various forms of stress, whereas Hsp105␤ is an alternatively spliced form of Hsp105␣ that is specifically produced following heat shock at 42°C (1-3). Hsp105␣ and Hsp105␤ suppress the aggregation of denatured proteins caused by heat shock in vitro, as does Hsp70, but refolding activity of these proteins is yet to be revealed (4). These proteins exist as complexes associated with Hsp70 and Hsc70 (a constitutive form of Hsp70) in mammalian cells (5,6) and suppress the chaperone activity of Hsc70 in vitro and in vivo (4,7). Furthermore, Hsp105␣ and Hsp105␤ are phosphorylated at Ser 509 by protein kinase CK2 (CK2) 1 in vitro and in vivo, and the CK2-mediated phosphorylation modulates the inhibitory effect of Hsp105␣ on Hsp70/Hsc70 chaperone activity (7). Recently, Hsp105␣ and Hsp105␤ were suggested to function as a substitute for Hsp70 family proteins to suppress the aggregation of denatured proteins in cells under severe stress, in which the cellular ATP level decreases markedly (8).
The HSP70 family is a major and well characterized group of heat shock proteins. Several different species of HSP70 family proteins are present in different compartments of eukaryotic cells and play important roles as molecular chaperones that prevent the irreversible aggregation of denatured proteins and assist folding, assembly, and translocation across the membrane of cellular proteins (9,10). The chaperone activity of Hsp70/Hsc70 relies on its ability to bind to short exposed hydrophobic stretches of polypeptide substrates in an ATP-regulated fashion. The ATP-bound Hsp70/Hsc70 exhibits low affinity and fast exchange rates for substrate, whereas the ADPbound form has high affinity and slow exchange rates for substrate (11)(12)(13)(14). Conversion of ATP-bound Hsp70/Hsc70 to the ADP-bound form is induced by its intrinsic ATPase activity, which is facilitated by co-chaperones of the HSP40 family (15). Many proteins have been identified as regulators of Hsp70/ Hsc70-mediated refolding of denatured proteins (16 -20). Hip stabilizes the ADP-bound form of Hsp70/Hsc70 and prevents the ATP-ADP cycle of Hsp70/Hsc70 (16). BAG-1 inhibits the chaperone activity of Hsp70/Hsc70 through the promotion of the dissociation of ADP from Hsp70/Hsc70 (17,18). CHIP suppresses the reaction cycle of Hsp70/Hsc70 by preventing the binding of ATP or inhibiting the hydrolysis of ATP (19,20).
The predicted secondary structure of Hsp105␣ and Hsp105␤ is composed of an N-terminal ATP-binding, a ␤-sheet, a loop, and a C-terminal ␣-helical domain, similar to those of HSP70 family proteins (2,3). The ␤-sheet domain of Hsp105␣ and Hsp105␤ binds denatured proteins such as Hsp70/Hsc70 (8). However, although the ATP-binding domain of Hsp105␣ and Hsp105␤ is conserved among HSP70 family proteins, the ATP binding of the domain in HSP105 family proteins has not been elucidated. Furthermore, although Hsp105␣ suppresses the chaperone activity of Hsp70/Hsc70 (4, 7), the precise mechanism of the suppression has not been clarified yet. In the present study, we examined the mechanisms by which Hsp105␣ regulates Hsc70 chaperone activity and revealed that Hsp105␣ suppresses the chaperone activity of Hsc70 by inhibiting the ATPase activity of Hsc70 with the concomitant appearance of Hsp105 ATPase activity.

EXPERIMENTAL PROCEDURES
Plasmids-Expression plasmids for His-tagged mouse Hsp105␣ and deletion mutants of Hsp105␣ in Escherichia coli have been described previously (4,7). To construct an expression plasmid (pTrcHis70) for His-tagged human Hsc70 in E. coli, human Hsc70 cDNA derived from the plasmid pHSC7 (21) was subcloned into XhoI-KpnI sites of the expression vector pTrcHisA (RIKEN Gene Bank, Ibaraki, Japan). For the construction of deletion mutants of Hsc70, a PCR was performed with pTrcHis70 as the template and specific 5Ј end-phosphorylated primers (underlining indicates the additional KpnI site): His-Hsc70N2 (1-501 amino acids), 5Ј-GGGGTACCAGATGTCCAAGGGACCTGCA-3Ј and 5Ј-GGGGTACCTCAAATCTTGTTCTCTTTTCCCGT-3Ј; His-Hsc-70C1 (509 -646 amino acids), 5Ј-GGGGTACCGTTTGAGCAAGGAAG-AC-3Ј and 5Ј-AATCTTCTCTCATCCGCC-3Ј; His-Hsc70C2 (393-646 * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to T. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Protein Purification-His-tagged proteins were purified by Ni 2ϩ -chelating agarose column chromatography (Invitrogen) followed by Mono Q anion-exchange column chromatography (Amersham Biosciences) (4, 7). Among a series of deletion mutants of Hsp105␣, only His-Hsp105C3 (see Fig. 1A) was purified by the Ni 2ϩ -chelating agarose column chromatography as the protein was tightly bound to and hardly eluted from the Mono Q column. To purify the GST-tagged mouse Hsp105␣, the transformant containing pGEX-105 was grown at 37°C in LB medium with 100 g/ml ampicillin until the A 600 reached 0.5. After a 3-h treatment with 0.3 mM isopropyl-␤-D(-)-thiogalactopyranoside at 37°C, the cells were ruptured by sonication, and the lysate was loaded onto a glutathione-Sepharose 4B column (Amersham Biosciences) equilibrated with phosphate-buffered saline (PBS). The column was washed with PBS, and bound protein was eluted with an elution buffer containing 50 mM Tris-HCl, pH 8.0, and 10 mM glutathione (reduced form). To remove the GST tag from GST-Hsc70K71A, 50 g of GST-Hsc70K71A was incubated with 10 units of ProScisstion protease (Amersham Biosciences) in 50 l of cleavage buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT) at 5°C for 24 h. Then, the reaction mixture was incubated with 20 l of glutathione-Sepharose 4B beads (50% in cleavage buffer) at 4°C for 1 h, and the unbound fraction that contains Hsc70K71A was collected. FIG. 2. Hsp105␣ mutants interacting with Hsc70 suppress the Hsc70 chaperone activity. Luciferase (164 nM) was incubated with Hsc70, Hsp40, and Hsp105␣ or a deletion mutant (2 M each) at 42°C for 30 min. Then, to 10-l aliquots, rabbit reticulocyte lysate was added at 40%, and the mixture was incubated further at 25°C for 30 min. Luciferase activity was assayed, and the relative activity of luciferase is expressed as a percentage of that of the control with Hsc70/Hsp40. Each value represents the means Ϯ S.D. from four independent experiments. Statistical significance was determined with the unpaired Student's t test. *, p Ͻ 0.05. BSA, bovine serum albumin. N, Hsp105N.
Purified proteins were separated by SDS-PAGE, and Coomassie Brilliant Blue-stained bands were quantified by densitometry. The concentration of proteins was estimated with bovine serum albumin as a standard.
In Vitro Pull-down Assay-The interaction between Hsc70 and deletion mutants of Hsp105␣ was analyzed by pull-down assay. His-tagged Hsp105␣ or the deletion mutant (20 M) was incubated with Hsc70 (20 M) in 50 l of a binding buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5 mg/ml bovine serum albumin at 4°C for 1 h. Then, the reaction mixtures were incubated with 10 l of Ni 2ϩ -chelating agarose at 4°C for 1 h, centrifuged, and washed several times with the binding buffer. Bound proteins were eluted with an elution buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 500 mM imidazole, separated by SDS-PAGE, and detected by Western blotting using anti-Hsp70 (Sigma) and anti-PentaHis antibodies (Qiagen).
The interaction between Hsp105␣ and deletion mutants of Hsc70 was determined by GST-pull down assay. His-tagged Hsc70, or the deletion mutant (20 M) was incubated with GST-Hsp105␣ (20 M) in 50 l of PBS at 4°C for 1 h, and then the mixtures were incubated with 10 l of glutathione-Sepharose 4B beads (50% in PBS) at 4°C for 1 h. The beads were washed several times with PBS, and bound proteins were eluted with a buffer containing 50 mM Tris-HCl, pH 8.0, and 10 mM glutathione, separated by SDS-PAGE, and detected by Western blotting using anti-GST (Amersham Biosciences) and anti-PentaHis antibodies.
Protein Refolding Assay-The protein refolding assay was conducted as described previously (4,7). Briefly, luciferase (164 nM) was incubated with Hsc70, Hsp105␣, and/or their mutants (2 M each) in a buffer containing 25 mM Hepes-KOH, pH 7.5, 50 mM KCl, 5 mM MgCl 2 , 5 mM DTT, and 2 mM ATP at 42°C for 30 min. To aliquots (10 l) of the reaction mixtures was added 20 l of reactivation buffer containing 25 mM Hepes-KOH, pH 7.5, 50 mM KCl, 5 mM MgCl 2 , 5 mM DTT, 2 mM ATP, 10 mM phosphocreatine, 3.5 units of creatine kinase, and 60% rabbit reticulocyte lysate. After incubation at 25°C for a predetermined period, luciferase activity was assayed using a luminometer after mixing an aliquot of the reaction mixture with 50 l of a luciferase assay solution (Promega).
Analysis of ATP Hydrolysis and ATPase Activity-ATP hydrolysis was determined as described previously (4)

Domains Necessary for the Interaction between Hsp105␣ and
Hsc70 -To elucidate how Hsp105␣ regulates the Hsc70 chaperone system, we first examined the interaction between Hsc70 and Hsp105␣ deletion mutants (Fig. 1A). A series of His-tagged Hsp105␣ deletion mutants was incubated with Hsc70, and pull-down assays were performed using Ni 2ϩ -chelating beads. Hsp105⌬L and Hsp105⌬C5 interacted with Hsc70 the same as wild-type Hsp105␣, whereas Hsp105⌬␤ and Hsp105⌬␤L lacking the ␤-sheet domain failed to interact with Hsc70. Furthermore, Hsc70 did not interact with Hsp105N2 or Hsp105C3, which lacked an ␣-helix and ATP-binding domain, respectively. These results indicated that all domains of Hsp105␣ except the loop are essential for the interaction between Hsp105␣ and Hsc70.
Next, we examined the interaction between Hsp105␣ and Hsc70 deletion mutants (Fig. 1B). A series of His-tagged Hsc70 deletion mutants were incubated with Hsp105␣, and pull-down assays were performed using Ni 2ϩ -chelating beads. Hsc70 deletion mutants lacking the N-terminal ATP-binding, ␤-sheet, or C-terminal ␣-helix domain did not interact with Hsp105␣, suggesting that all domains of Hsc70 are necessary for the binding to Hsp105␣.
Interaction of Hsp105␣ with Hsc70 Is Required for the Suppression of Hsc70 Chaperone Activity-We next examined the suppressive effect of Hsp105␣ and its mutants on Hsc70 chap-  Hsp105N. B, Hsp105␣ or its deletion mutant (2 M each) was incubated with or without Hsc70 (1 M) and Hsp40 (0.5 M) in buffer containing 500 M [␥-32 P]ATP (0.1 mCi/mmol). Then, ATP hydrolysis was determined. Among deletion mutants, as the Hsp105␣ mutant lacking an ATP-binding domain (Hsp105C3) could not be purified by MonoQ column chromatography due to its tight adsorption to the column, the Hsp105C3 preparation that still contained some bacterial proteins was not used for the measurement of ATP hydrolysis. Each value represents the means Ϯ S.D. from three independent experiments. Statistical significance was determined with the unpaired Student's t test. *, p Ͻ 0.01; **, p Ͻ 0.05. erone activity. Luciferase was incubated with Hsc70, Hsp40, and Hsp105␣ deletion mutants at 42°C for 30 min, and luciferase activity was assayed after the addition of rabbit reticulocyte lysate (Fig. 2). Hsp105⌬L and Hsp105⌬C5, which lacked the loop and the C-terminal 5 amino acids, respectively, and were able to interact with Hsc70, suppressed Hsc70 chaperone activity similar to Hsp105␣. In contrast, the mutants that were unable to interact with Hsc70 did not suppress the chaperone activity of Hsc70. Thus, the direct interaction of Hsp105␣ with Hsc70 seemed to be necessary for the suppression of Hsc70 chaperone activity.
Hsp105␣ That Interacts with Hsc70 Enhances ATP Hydrolysis in the Reaction with Hsc70, Hsp40, and Hsp105␣-We have shown that hydrolysis of ATP increases when Hsp105␣ is added to the reaction with Hsc70 and Hsp40 (4). Then, we next examined the effect of Hsp105␣ deletion mutants on ATP hydrolysis in the reaction with Hsc70 and Hsp40 (Fig. 3). Hsc70 displayed a low basal rate of ATP hydrolysis, which was enhanced ϳ4-fold by Hsp40 (Fig. 3B). On the other hand, Hsp105␣ and its deletion mutants did not show any intrinsic ATPase activity (Fig. 3A). However, ATP hydrolysis in the reaction with Hsc70, Hsp40, and Hsp105␣ or the deletion mutant of Hsp105⌬L or Hsp105⌬C5 was enhanced ϳ2-fold when compared with that in the reaction with Hsc70 and Hsp40 (Fig.  3B). The Hsp105␣ mutants that did not interact with Hsc70 and suppress Hsc70 chaperone activity did not affect the hydrolysis of ATP in the reaction with Hsc70 and Hsp40. Thus, Hsp105␣ that interacted with Hsc70 seemed to enhance ATP hydrolysis in the reaction with Hsc70 and Hsp40. These findings suggest either that Hsp105␣ enhances the ATPase activity of Hsc70 or that Hsc70 and Hsp40 induce ATPase activity of Hsp105␣.
Hsp105␣ Suppresses ATPase Activity of Hsc70 -Although Hsp105␣ contains an ATP-binding consensus sequence similar to that of HSP70 family proteins, no ATPase activity of Hsp105␣ has been detected yet. Then, we first examined the possibility that Hsp105␣ enhances the ATPase activity of Hsc70 (Fig. 4). When Hsc70 was incubated with ATP in the absence of K ϩ , Hsc70 existed predominantly in the ATP-bound form, whereas in the presence of K ϩ , Hsc70-ATP was hydrolyzed to ADP due to the intrinsic ATPase activity of Hsc70. The hydrolysis of ATP was significantly enhanced by the addition of Hsp40, consistent with the stimulation of Hsc70 ATPase activity by Hsp40 (15). The addition of Hsp105␣, either with or without Hsp40, suppressed the hydrolysis of Hsc70-bound ATP in a dose-dependent manner (Fig. 4A). Furthermore, when the effect of Hsp105␣ deletion mutants on the hydrolysis of Hsc70bound ATP was examined, Hsp105⌬L and Hsp105⌬C5 that interacted with Hsc70 were found to suppress the hydrolysis of Hsc70-bound ATP, similar to Hsp105␣. However, the mutants that did not interact with Hsc70 did not suppress the hydrolysis (Fig. 4B). These findings suggested that Hsp105␣ did not enhance the ATPase activity of Hsc70 but rather suppressed Hsc70 ATPase activity by interacting with Hsc70.
ATPase Activity of Hsp105␣ Is Induced by Hsc70 -We examined the second possibility, that Hsc70 and Hsp40 induce ATPase activity of Hsp105␣. When Hsp105␣ was incubated with ATP in the absence of K ϩ , Hsp105␣ existed predominantly as the ATP-bound form, and Hsp105␣-bound ATP was not hydrolyzed to ADP even in the presence of K ϩ (Fig. 5A). However, Hsp105␣-bound ATP was converted to ADP by Hsc70, either with or without Hsp40, in a dose-dependent manner, but not by Hsp40 alone (Fig. 5, A and B).
Then, to determine whether the conversion of Hsp105␣bound ATP to ADP by Hsc70 is due to the ATPase activity of Hsc70, we prepared an Hsc70 mutant that was defective in ATP binding. The Lys residue at position 71 of human Hsc70, which is essential for hydrolysis of ATP, was substituted with Ala to yield Hsc70K71A (23). Hsc70K71A did not bind ATP (Fig. 6A) but interacted with Hsp105␣ (Fig. 6B). When ATPbound Hsp105␣ was incubated with Hsc70WT or Hsc70K71A, hydrolysis of Hsp105␣-bound ATP was observed in the presence or absence of K ϩ , and the hydrolysis was not significantly affected by Hsp40 (Fig. 6C). Furthermore, although Hsc70 did not show any ATPase activity in the absence of K ϩ , Hsc70WT also enhanced the hydrolysis of Hsp105␣-bound ATP in the absence of K ϩ . These findings suggest that the ATPase activity of Hsc70 is not necessary for the hydrolysis of ATP bound to Hsp105␣, whereas the ATPase activity of Hsp105␣ is induced by interaction with Hsc70.

DISCUSSION
Hsp105␣ associates with Hsp70/Hsc70 (5, 6) and suppresses its chaperone activity in mammalian cells (4,7). The mechanism by which Hsp105␣ regulates the chaperone activity of Hsp70/Hsc70, however, had not been elucidated. Here, we demonstrated that Hsp105␣ suppresses the ATPase activity of the Hsc70 chaperone, with the concomitant appearance of Hsp105␣ ATPase activity.  (20 M) in 50 l of binding buffer for 1 h at 4°C, and then a pull-down assay using Ni 2ϩchelating agarose was performed. Proteins bound to the beads were eluted, separated by SDS-PAGE, and detected by Western blotting using anti-Hsp70 and anti-PentaHis antibodies. C, [␣-32 P]ATP-bound Hsp105␣ (1 M) was incubated with Hsc70WT or Hsc70K71A (1 M each) in the presence or absence of Hsp40 (0.5 M) in buffer containing or not containing K ϩ at 25°C for 10 min, and the conversion of Hsp105␣-bound ATP to ADP was analyzed by thin-layer chromatography. K, Hsc70K71A.